Lanterna Rally

Author: Mariam Sarvi

Publisher:

Published: 2014

Total Pages: 416

ISBN-13:

Embryonic stem cells (ESCs) are pluripotent cells capable of indefinite self renewal in vitro while maintaining the ability to differentiate into cell types of all three germ layers. ESCs are outstanding options of in vitro cell models for regenerative medicine, functional genomics, human developmental biology and drug discovery study. Stem cell research is among the most promising fields of biotechnology, and which provides the potential of developing novel approaches to repair or replace damaged tissues and cells. The present day, exponentially growing effort of stem cell research emphasizes a major need for convergence of more efficient and appropriate laboratory technologies to sustain the growth, proliferation and differentiation potential of stem cells.Although so far, a variety of conventional bioreactors with different configurations (such as spinner flasks, rotary, perfusion bioreactor, etc.) have been designed and adapted for stem cell expansion and differentiation, bioreactors can be disadvantageous in bench-top research because they need large space, consume huge amount of reagents and need more time to operate and maintain (sterilizing, cleaning, assembling, and disassembling of the bioreactor components). The requirements for costly equipment and generating shear stress due to the fluid flow, and the lack of physical similarity between microenvironments of bioreactor and actual cell microenvironment, make using bioreactors undesirable.In tissue engineering, micro-scale technology is an approach that combines micro-techniques with materials science and surface engineering, and results in a profound exploration of the microenvironment where cells are embedded. These technologies are able to address some of the limitations imposed by conventional tissue engineering methods. Indeed, developing successful novel small-scale technologies for in vitro cultivation of different types of cells can assist in increasing our knowledge on conditions that control stem cell growth and differentiation and organ development. In fact, small scale bioreactors are miniaturized versions of conventional bioreactors, where high- throughput cell based assays can be carried out at low cost compared with their macro-scale counter- parts.The first aim of this thesis was to develop a disposable miniaturized bioreactor through a novel and inexpensive method for effective stem cell proliferation. To this end, an effective surface functionalization method was developed for enhancing the biocompatibility of a PDMS surface that is protein resistant while facilitating cell proliferation (expansion) and maintaining the pluripotency potential of cells. The micro-bioreactor was fabricated in the form of a fixed bed bioreactor with a microchannel reactor bed. The microchannel was functionalized to enable cell adhesion and resistance to protein adsorption. The functionalized surface was found to be biocompatible with cancer and embryonic stem cells (ESCs), and while facilitating cell proliferation.Differentiation of ESCs into a variety of cell types is an important characteristic of these types of cells, which is commonly achieved in vitro by spontaneously self-assembling in low adhesion culture conditions into 3D cell aggregates called embryoid bodies (EBs). Formation of EBs that simulates many of the characteristics of early embryonic development is considered as a vital step to induce differentiation of stem cells. Formation of three dimensional configurations of ESCs as EBs provides possibilities to mechanistically study early differentiation events of pluripotent cells. In fact, EB formation is of paramount importance for in vitro investigation of the embryonic development and differentiation of both the mouse and human ES cells.The second aim of this thesis was to introduce a novel concept of a miniaturized bioreactor made of liquid marble (LM). A novel application of liquid marbles for formation of embryoid bodies (EBs) was then presented. This study showed that the confined internal space of liquid marble, along with the porous and non-adhesive property of the highly hydrophobic liquid marble shell, can facilitate the formation of uniform EBs inside the liquid marbles. The efficiency of liquid marble-born-EBs compared to the liquid suspension (LS) technique as the chosen control method in terms of cell viability and EB uniformity revealed that cells in liquid marble are more viable than those in suspension. Measuring EB size distribution as an indicator of uniformity also confirmed that EBs obtained by the LM are morphologically more uniform and of a narrower size distribution compared to those formed in LS. The feasibility of using liquid marble bioreactors for cardiomyocyte differentiation of mouse ES (mES) cells after formation of EBs inside the liquid marble was further investigated. The results demonstrated that ES cells can differentiate into myocyte cells through the liquid marble as a facile, cost effective, and straightforward method. We proposed for the first time that liquid marbles greatly contribute to ES cardiac differentiation, which provides a new technology platform for ES biology and genetic studies.It is worth mentioning that although the majority of our knowledge in modern biology has been provided by classical two dimensional (2D) cell culture techniques, the most common negative aspects of these systems is deficiency of support from extra-cellular matrix, which represents an important role for cell growth and development. It is now well accepted that cells reside, proliferate, and differentiate in complex 3D microenvironments. The concept of using three dimensional (3D) biodegradable scaffolds as alternatives for extracellular matrix (ECM), which more closely reform cells' native structure, is an interesting area of study in current tissue engineering. Because of their unique function, stem cells need to reside in the specialized, 3D microenvironment that surrounds them in native tissues.The third section of this thesis (chapter 5) focuses on the investigating of the feasibility of forming embryoid bodies using a novel hydrogel as bioreactor embedding material. This hydrogel is porous and biodegradable and is prepared by modifying hydroxypropylcellulose (HPC), with bio-functional methacrylates (MA). Observation of EB formation inside hydrogel implied that the stem cells attached and penetrated to the pores, and proliferated well, while forming uniform EBs. Uniformity of EBs formed inside hydrogel, compared with those formed via liquid suspension (LS) method, as one of the most widely used EB formation techniques. It was observed that porous hydrogel allows the formation of more homogeneous EBs. It was found that cells inside hydrogel-born EBs are more viable compared to those formed in LS method. Expression of germ markers via quantitative PCR and immunostaining confirmed that the hydrogel-born EBs had expressed 3 germ layers with further in vitro differentiation potential. These EBs were allowed to differentiate further in hydrogel, where positive immunostaining of different cardiac markers and observation of beating EBs showed the potential of EBs to further differentiate into cardiac cells lineage.In summary, this thesis first presents a novel, facile and cost effective method via surface bio- functionalization of PDMS bioreactor for better stem cell adhesion and proliferation, and later introduces two novel methods to prepare bioreactor material, namely liquid marble and porous hydrogel (HPC-MA) for formation of embryoid bodies, which is considered as a critical step for in vitro differentiation in ESCs.